Hantaviruses are rodent-borne agents that cause hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome (HPS) in humans (de et al., 2002). Annually, around 150,000 cases of HFRS are reported worldwide, caused by Hantaan (HTNV) and Seoul (SEOV) hantaviruses in Asia and by Puumala (PUUV), Dobrava (DOBV), and Saaremaa (SAAV) hantaviruses in Europe (Klingstrom et al., 2004). Hantavirus was first identified in the United States in 1993. HPS is a deadly disease from rodents. Although rare, Although rare, the consequences of getting it are serious. Early symptoms include fatigue, fever and muscle aches. There may also be headaches, dizziness, chills, and abdominal problems, such as nausea, vomiting, diarrhea, and abdominal pain. The major HPS symptoms may not appear until the illness becomes life-threatening. Rodent control in and around the home remains the primary strategy for preventing hantavirus infection (CDC Hantaviruses). Hantaviruses can also cause human hemorrhagic fever with renal syndrome (HFRS) (Lednicky, 2003).
Increased numbers of rodents in the household is the strongest risk factor for infection. Among documented U.S. cases of HPS, patients with potential occupational exposures have included grain farmers, an extension livestock specialist, field biologists, and agricultural, mill, construction, utility and feedlot workers (CDC Hantaviruses).
Hantaviruses contain a three-segmented ssRNA-genome of negative polarity encoding four proteins: the nucleocapsid protein (N), the envelope proteins (G1 and G2), and the RNA dependent RNA polymerase (Klingstrom et al., 2004).
4. Microbial Pathogenesis
Rodents (primarily deer mice) carry hantaviruses that cause hantavirus pulmonary syndrome in human. Rodents shed the virus in their urine, droppings, and saliva. The virus is mainly transmitted to people when they breathe in air contaminated with the virus. Functional impairment of vascular endothelium is central to the pathogenesis of HPS, and a myocardial depressant may contribute significantly to the mortality of this disease. Hantaviruses can infect endothelial cells, macrophages and dendritic cells. The mechanism of inflammatory cell recruitment in the lungs of HPS patients may result from specific attraction and adherence of a selective population of inflammatory cells to an activated pulmonary microvascular endothelium (CDC Hantaviruses).
5. Host Ranges and Animal Models
n the US, primarily deer mice, along with cotton rats and rice rats in the southeastern states and the white-footed mouse in the Northeast, carry hantaviruses that cause hantavirus pulmonary syndrome (CDC Hantaviruses).
6. Host Protective Immunity
Klingström et al. reported that the strong Th2-type of immune response induced by Alum against rDOBV N did not induce protection in mice (Klingstrom et al., 2004).
II. Vaccine Related Pathogen Genes
1. ANDVsSgp1
Gene Name :
ANDVsSgp1
Sequence Strain (Species/Organism) :
Andes orthohantavirus
Molecule Role Annotation :
Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN, ANDVsSgp1) or glycoproteins (AdG(N) and AdG(C)) were constructed . When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. No vaccinated animal died, and there were no obvious clinical signs of disease (Safronetz et al., 2009).
>AAC54579.1 envelope glycoprotein G1, partial [Sin Nombre orthohantavirus]
FLVVLTTATAGLTRNLYELKIECPHTVGLGQGYVTGSVETTPVLFSQVADLKIESSCNFDLHVPATTTQK
YNQVDWTKKSSTTENTNAGAS
Molecule Role :
Protective antigen
Molecule Role Annotation :
Study used a deer mouse infection model to test the protective efficacy of genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological responses in BALB/c mice. Protective epitopes were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus G1 glycoprotein antigen; the nucleocapsid gene also was protective (Bharadwaj et al., 2002).
Molecule Role Annotation :
Seoul virus (SEOV) is one of the four known hantaviruses causing hemorrhagic fever with renal syndrome (HFRS). A replication-competent recombinant canine adenovirus type 2 expressing the Gc protein of SEOV (rCAV-2-Gc) in BALB/c mice induced complete protection against a intensive infectious challenge with ~1,000 50% infective doses (ID50) for SEOV strain CC-2 (Yuan et al., 2010).
>gi|2801762|gb|AF042137.1|AF042137 Andes virus Case T M segment polyprotein gene, partial cds
AAGGAAATACCATTTCTGGATATAAAAGAATGATGGCAACAAAAGATTCATTCCAATCGTTTAACTTAAC
AGAACCTCACATCACAGCAAATAAGCTTGAATGGATTGACCCAGATGGAAATACAAGAGACCATGTAAAT
CTTGTCTTAAATAGAGATGTTTCATTT
Molecule Role Annotation :
Passive transfer of spleen cells from mice immunized with rNP (HTNVsSgp1) conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD50 (survival rate: 43%) or 4 LD50 (survival rate: 43%) (Yoshimatsu et al., 1993).
>AAB02907.1 M polyprotein, partial [Hantaan orthohantavirus]
TPLTPVWNDNAHGVGSVPMHTDLELDFSLTSSSKYTYRRKLTNPLEEAQSVDLHIEIEEQTIGVDVHALG
HWFDGRLNLKTSFHCYGACTKYDYPWHTAKCHYERDYQ
Molecule Role :
Protective antigen
Molecule Role Annotation :
Researchers vaccinated hamsters with a vaccinia virus-vectored vaccine expressing the M and the S segments of Hantaan (HTN) virus and challenged them with HTN, Seoul (SEO), or Puumala (PUU) virus. Study found that vaccinated hamsters, challenged with HTN or SEO virus, were neither viremic nor had evidence of virus in their lungs or kidneys (Chu et al., 1995).
Molecule Role Annotation :
To investigate the ability of recombinant N (rN, nucleocapsid proteins) from different hantaviruses to elicit cross-protection, we immunized bank voles with rN from Puumala (PUUV), Topografov (TOPV), Andes (ANDV), and Dobrava (DOBV) viruses and subsequently challenged them with PUUV. All animals immunized with PUUV (PUUVsSgp1) and TOPV rN were completely protected (de et al., 2002).
Molecule Role Annotation :
Study compared the immunogenicity and protective efficacy of recombinant DOBV nucleocapsid protein (rDOBV N, S) given with Alum or Freund's as adjuvant, or PBS, in C57/BL6 mice. Mice receiving rDOBV N with Freund's adjuvant were protected from challenge (75% protected) (Klingstrom et al., 2004).
>CAB42097.1 N protein [Topografov hantavirus]
MSNLKDIQDEITRYEQQLIVARQKLRDAEKTVEVDPDDVNKNTLQARRQTVSALEDKLADFKRQLADHVS
RQKMDEKPSDPTGIEPDDHLKERSSLRYGNVLDVNAIDIEEPSGQTADWFTIGVYIVSFTLPIILKALYM
LSTRGRQTVKENKGTRIRFKDDSSFEDVNGIRRPKHLYVSMPTAQSTMKAEELTPGRFRTIVCGLFPAQI
QARNIMSPVMGVIGFSFFVKDWPERIKNFLESKCPFIKPEVKPGAPAGEADFLSRNQIYFMRRQEVLEDN
HIPDIDKLLEYASSGDPTAPDSIESPYAPWVFACAPDRCPPTCIYIAGMAELGAFFSILQDMRNTIMASK
TVGTAEEKLKKKSSFYQSYLRRTQSMGIQLDQRIILLYMVEWGKEMVDHFHLGDDMDPDLRNLAQSLIDQ
KVKEISNQEPLKI
Molecule Role :
Protective antigen
Molecule Role Annotation :
To investigate the ability of recombinant N (rN, nucleocapsid proteins) from different hantaviruses to elicit cross-protection, we immunized bank voles with rN from Puumala (PUUV), Topografov (TOPV), Andes (ANDV), and Dobrava (DOBV) viruses and subsequently challenged them with PUUV. All animals immunized with PUUV and TOPV rN were completely protected (de Carvalho Nicacio et al., 2002).
>NP_941975.1 nucleocapsid protein [Sin Nombre orthohantavirus]
MSTLKEVQDNITLHEQQLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLGELKRELADLIA
AQKLASKPVDPTGIEPDDHLKEKSSLRYGNVLDVNSIDLEEPSGQTADWKSIGLYILSFALPIILKALYM
LSTRGRQTIKENKGTRIRFKDDSSYEEVNGIRKPRHLYVSMPTAQSTMKADEITPGRFRTIACGLFPAQV
KARNIISPVMGVIGFSFFVKDWMERIDDFLAARCPFLPEQKDPRDAALATNRAYFITRQLQVDESKVSDI
EDLIADARAESATIFADIATPHSVWVFACAPDRCPPTALYVAGMPELGAFFAILQDMRNTIMASKSVGTS
EEKLKKKSAFYQSYLRRTQSMGIQLDQKIIILYMSHWGREAVNHFHLGDDMDPELRELAQTLVDIKVREI
SNQEPLKL
Molecule Role :
Protective antigen
Molecule Role Annotation :
Study used a deer mouse infection model to test the protective efficacy of genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological responses in BALB/c mice. Protective epitopes were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus G1 glycoprotein antigen; the nucleocapsid gene (SNVsSgp1) also was protective (Bharadwaj et al., 2002).
Description:
Nonreplicating (E1− E3−) Ad vectors expressing ANDV proteins were constructed using the AdMax HI-IQ system (Microbix, Toronto, Canada). The ANDV GN, GC, GPC, and N open reading frames were PCR amplified from plasmids containing the appropriate cDNAs derived from a Chilean ANDV isolate, strain 9717869. AdEmpty was constructed using plasmid pDC316(io) containing no ANDV sequences. These pDC316(io)-based ANDV plasmids were cotransfected with plasmid pBHGloxΔE1,3Cre, containing the remainder of the Ad5 genomic plasmid, into 293 IQ cells. Supernatants were collected after 6 to 8 days, and the presence of Ad vectors expressing the ANDV proteins (designated AdN, AdGN, AdGC, and AdGPC) was confirmed by immunoprecipitation. Ad vectors were plaque purified, propagated in large-scale infections in 293 IQ cells, and purified using standard CsCl gradient methods (Safronetz et al., 2009).
Vaccination Protocol:
Syrian golden hamsters (Mesocricetus auratus) (4- to 6-week-old males; Charles River, Pointe Claire, Canada) were group housed in microisolator units situated in the biosafety level 4 area of the National Microbiology Laboratory, Public Health Agency of Canada. Prior to vaccine experiments, the 50% lethal dose (LD50) for i.p. injections of ANDV in hamsters was established by inoculating anesthetized animals with 0.8 to 80,000 FFU (using 10-fold dilutions) of ANDV. Hamsters were monitored twice daily for clinical signs of illness according to an approved scoring sheet (ruffled fur, lethargy, inappetence, and labored breathing). For vaccination, hamsters were anesthetized with isoflurane, a preimmunization blood sample was collected, and the animals were immunized with the Ad vectors using 108 (293 cell) PFU of each vector diluted in 100 μl phosphate-buffered saline at two sites in the hind-leg musculature (Safronetz et al., 2009).
Challenge Protocol:
After 28 days, a second blood sample was collected and hamsters were challenged with ANDV by i.p. injection of 100 LD50s (equivalent to 154 FFU). Hamsters were examined twice daily for signs of illness. Survivors were monitored for 40 to 45 days and then anesthetized and exsanguinated via cardiac puncture (Safronetz et al., 2009).
Efficacy:
Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN, ANDVsSgp1) or glycoproteins (AdG(N) and AdG(C)) were constructed . When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. No vaccinated animal died, and there were no obvious clinical signs of disease (Safronetz et al., 2009).
Chimaeric hepatitis B virus (HBV) core particles were constructed to carry defined fragments of the Puumala virus nucleocapsid protein. Puumala is a European hantavirus (Ulrich et al., 1998).
pWRG/HTN-M(x), a customized plasmid built from pWRG7077 (Custer et al., 2003)
f. Preparation
An expression plasmid, pWRG/AND-M, was constructed that contains the full-length M genome segment of Andes virus (ANDV), a South American hantavirus (Custer et al., 2003).
g.
Macaque Response
Vaccine Immune Response Type:
VO_0000286
Efficacy:
Rhesus macaques vaccinated by gene gun with pWRG/AND-M developed remarkably high levels of neutralizing antibodies that not only neutralized ANDV (a South American hantavirus) but also cross-neutralized other HPS-associated hantaviruses, including Sin Nombre virus (Custer et al., 2003).
4. Dobrava-Belgrade virus S nucleocapsid protein vaccine
e. Gene Engineering of
S nucleocapsid protein from Dobrava-Belgrade virus
Type:
Recombinant protein preparation
Description:
His-tagged rDOBV N and recombinant mouse dihydrofolate reductase (rDHFR) was prepared. Freund’s complete adjuvant (FCA) or incomplete (FIA) adjuvant (Sigma, St. Louis, MO) or PBS. A total of 50 μg of recombinant protein was mixed with Alum and FCA/FIA, according to the manufacturers’ descriptions, or PBS, in a total volume of 200 μl per dosage (Klingstrom et al., 2004).
Vaccination Protocol:
A total of 50 μg of recombinant protein was mixed with Alum and FCA/FIA, according to the manufacturers’ descriptions, or PBS, in a total volume of 200 μl per dosage. All immunizations and boosters were administered subcutaneously. rDOBV N or rDHFR in Alum, FCA or PBS were administered at day 0. At days 21 and 92, mice were boosted with rDOBV N or rDHFR in Alum, FIA or PBS (Klingstrom et al., 2004).
Challenge Protocol:
At day 118, all mice were challenged with 10 mouse ID50 of DOBV and 21 days later, all mice were sacrificed. Serum, plasma, and EDTA-blood were drawn at the time points indicated below (Klingstrom et al., 2004).
Efficacy:
Study compared the immunogenicity and protective efficacy of recombinant DOBV nucleocapsid protein (rDOBV N, S) given with Alum or Freund's as adjuvant, or PBS, in C57/BL6 mice. Mice receiving rDOBV N with Freund's adjuvant were protected from challenge (75% protected) (Klingstrom et al., 2004).
Host Gene Response of
Il2
Gene Response:
Significantly higher levels of IL-2 producing cells were found in the group given rDOBV N with Alum as compared to the rDHFR vaccinated groups and PBS vaccinated groups. These results were found in mice peripheral blood mononuclear cells (PBMCs) 113 days after vaccination (Klingstrom et al., 2004).
Gene Response:
Significantly higher numbers of IL-4 producing cells were found in the groups given rDOBV N with Freund’s adjuvant as compared to rDOBV N with PBS. These reactions were found in peripheral blood mononuclear cells (PBMCs) 113 days after vaccination (Klingstrom et al., 2004).
Efficacy:
All of the hamsters that were vaccinated with pWRG/HTN-M were protected against infection as defined by an absence of a postchallenge N-specific antibody response. In addition, the pre- and postchallenge PRNT titers differed by ≤4-fold. In contrast, all of the negative control hamsters, whether they were vaccinated with pWRG7077 or remained unvaccinated, were infected, as evidenced by the development of N-specific antibodies and neutralizing antibodies postchallenge (Hooper et al., 2001).
Description:
N proteins from PUUV, TOPV, ANDV, and DOBV, carrying a polyhistidine tag to facilitate protein purification, were produced in Escherichia coli cells. The N ORF of PUUV, TOPV, and DOBV were cloned into the pQE-32 vector (Qiagen, Hilden, Germany), while the N ORF of ANDV was cloned into pRSET (R&D Systems Europe, Oxford, United Kingdom). An irrelevant protein, mouse dihydrofolate reductase (DHFR) (Qiagen), provided by the manufacturer and expressed in the same system, was used as a negative control protein (de et al., 2002).
Vaccination Protocol:
To asses the immunogenicity and protective capacity of the expressed rN proteins, 4- to 10-week-old bank voles, derived from a PUUV-free colony established with animals captured in Sweden, were immunized with purified PUUV, TOPV, ANDV, or DOBV rN or DHFR control protein. Bank voles were immunized three times with 50 μg of protein at intervals of 3 weeks. The animals were injected with protein emulsified in Freund's complete adjuvant, incomplete Freund's adjuvant, and phosphate-buffered saline (PBS), respectively (de et al., 2002).
Challenge Protocol:
Bank voles were challenged subcutaneously 2 weeks after the last immunization with approximately 20 50% infective doses of wild-type PUUV (strain Kazan) (de et al., 2002).
Efficacy:
To investigate the ability of recombinant N (rN, nucleocapsid proteins) from different hantaviruses to elicit cross-protection, we immunized bank voles with rN from Puumala (PUUV), Topografov (TOPV), Andes (ANDV), and Dobrava (DOBV) viruses and subsequently challenged them with PUUV. All animals immunized with PUUV (PUUVsSgp1) and TOPV rN were completely protected (de et al., 2002).
7. Recombinant DOBV nucleocapsid protein (rDOBV N)
Description:
Dobrava hantavirus nucleocapsid protein in Freund's adjuvant (Klingstrom et al., 2004).
e. Preparation
Recombinant DOBV nucleocapsid protein (rDOBV N) given with Alum or Freund's as adjuvant (Klingstrom et al., 2004).
f. Virulence
Not virulent
g. Description
Dobrava hantavirus (DOBV) causes a severe form of hemorrhagic fever with renal syndrome (HFRS). Currently there is no therapy or vaccine available for HFRS (Klingstrom et al., 2004).
h.
Mouse Response
Host Strain:
C57/BL6 mice
Vaccination Protocol:
His-tagged rDOBV N and recombinant mouse dihydrofolate reductase (rDHFR) was emulsified with Imject® Alum (Pierce, Rockford, IL), Freund’s complete adjuvant (FCA) or incomplete (FIA) adjuvant (Sigma, St. Louis, MO) or PBS. A total of 50 μg of recombinant protein was mixed with Alum and FCA/FIA, or PBS, in a total volume of 200 μl per dosage. All immunizations and boosters were administered subcutaneously. rDOBV N or rDHFR in Alum, FCA or PBS were administered at day 0. At days 21 and 92, mice were boosted with rDOBV N or rDHFR in Alum, FIA or PBS. At day 118, all mice were challenged with 10 mouse ID50 of DOBV and 21 days later, all mice were sacrificed. Serum, plasma, and EDTA-blood were drawn at the time points indicated below (Klingstrom et al., 2004).
Side Effects:
No side effects.
Challenge Protocol:
All vaccinated mice were subcutaneously challenged with 10 mouse ID50 DOBV at day 118. The challenge did not kill mice. Three weeks after challenge, the mice were sacrificed and serum tested for the presence of neutralizing antibodies (Klingstrom et al., 2004).
Efficacy:
The immunogenicity and protective efficacy of recombinant DOBV nucleocapsid protein (rDOBV N) given with Alum or Freund’s as adjuvant, or PBS, in C57/BL6 mice, were compared. All mice given Alum or Freund’s seroconverted as did 6/8 mice given rDOBV N with PBS. Reciprocal geometric mean total IgG-titers were 5380, 18,100, and 800, respectively, while the mean IgG1/IgG2a ratios were 17.5, 9.25, and 12, respectively. Furthermore, ELIspot assays showed higher levels of IL-4 producing peripheral blood mononuclear cells (PBMCs) in the group given Alum as compared to the other groups. Interestingly, only mice receiving rDOBV N with Freund’s adjuvant were protected from challenge (75% protected), indicating that the strong Th2-type of immune response induced by Alum against rDOBV N did not induce protection in mice (Klingstrom et al., 2004).
Vaccination Protocol:
Syrian hamsters were immunized with a single injection of VSVΔG/ANDVGPC (Brown et al., 2011).
Vaccine Immune Response Type:
VO_0000287
Challenge Protocol:
After immunization, the hamsters were challenged at 28, 14, 7, or 3 days postimmunization with a lethal dose of ANDV (Brown et al., 2011).
Efficacy:
The hamsters were fully protected against the disease; however, the mechanism of protection seems to differ depending on when the immunization occurs. Administration of the vaccine at 7 or 3 days before challenge also resulted in full protection but with no specific neutralizing humoral immune response, suggesting a possible role of innate responses in protection against challenge virus replication. Administration of the vaccine 24 h postchallenge was successful in protecting 90% of hamsters and again suggested the induction of a potent antiviral state by the recombinant vector as a potential mechanism (Brown et al., 2011).
Description:
The Gc protein expressed by rCAV-2-Gc in MDCK cells was evaluated by a SEOV-specific indirect IFA. MDCK cells grown on 15 mm glass coverslips in 12-well culture plates were infected with rCAV-2-Gc or CAV-2 at an m.o.i. of 20. After 48 h infection, the coverslips were rinsed once with PBS (pH 7.4), fixed with acetone for 10 min at room temperature and then reacted with rabbit anti-SEOV polyclonal antiserum and washed three times with PBS. The fixed monolayers were incubated at 37 °C for 30 min in a moist chamber with donkey anti-rabbit IgG labelled with fluorescence isothiocyanate (Amersham). The coverslips were rinsed three times with PBS. Cell monolayers that bound the antibody were covered with glycerine and examined for specific fluorescence under a Zeiss Axioplan fluorescence microscope. Expression of SEOV Gc in MDCK cells infected with rCAV-2-Gc was identified by Western blotting. rCAV-2-Gc-infected cell lysates were separated by 12 % SDS-PAGE, and the proteins transferred to nitrocellulose membrane (Pall Corporation) and probed with positive serum against SEOV and HRP-labelled goat anti-mouse IgG antibody (Sigma) (Yuan et al., 2010).
replication-competent recombinant canine adenovirus type 2
g. Immunization Route
Intramuscular injection (i.m.)
h.
Mouse Response
Host Strain:
BALB/c
Vaccination Protocol:
Mice were randomly assigned to four experimental groups (20 mice per group). Group I was intramuscularly inoculated once with 0.1 ml rCAV-2-Gc (108.0 p.f.u. ml–1); group II received 0.1 ml CAV-2 (108.2 p.f.u. ml–1) intramuscularly as a negative control; group III were inoculated intramuscularly with one dose of HFRS bivalent purified vaccine Youerjian (0.5 ml per dose; GuangDong HongMing Biological Science and Technology Co.) as a positive-control; and group IV were injected with 0.1 ml PBS as a negative control (Yuan et al., 2010).
Challenge Protocol:
At the end of the trial, all mice were injected intramuscularly with SEOV strain CC-2 diluted in 0.2 ml PBS. The challenge dose for each virus was 2000 p.f.u. This dose is 1000 50 % infective doses for SEOV. At 14 days after challenge, the mice were sacrificed by CO2 asphyxiation, as approved by the China Small Animal Protection Association. Pre- and post-challenge sera were evaluated for the presence of N-specific antibodies by ELISA and for the presence of neutralizing antibodies by FRNT. Detecting post-challenge N-specific antibody indicated that the mice were infected with the challenge virus (Yuan et al., 2010).
Efficacy:
eplication-competent recombinant canine adenovirus type 2 expressing the Gc protein of SEOV (rCAV-2-Gc) in BALB/c mice induced complete protection against a intensive infectious challenge with ~1,000 50% infective doses (ID50) for SEOV strain CC-2 (Yuan et al., 2010).
Description:
Cloned the G1 and G2 glycoprotein genes of SN virus strain CC107 into the CMV expression vector pCMVi (-H3) UBs (Bharadwaj et al., 1999 ). The M segment fragments 3' of the first fragment were prepared in a similar manner, such that each expression construct shared 100 nt of sequence at the 5' end with the 3' end of the fragment that preceded it. The coordinates of each of the ten glycoprotein fragments, designated M-CMV-A thorough -I. We also cloned the entire SN virus N gene in a single fragment in a separate expression construct. The same viral cDNA fragments were cloned into bacterial expression vectors to allow bacterial synthesis of the cognate antigens as fusion proteins, as described (Bharadwaj et al., 1997 ; Yamada et al., 1995 ) (Bharadwaj et al., 2002).
Vaccination Protocol:
We purified plasmid DNA with an endotoxin-free kit (EndoFree, Qiagen), and dissolved DNA to a concentration of 1 mg/ml in 0·9% NaCl. Five to twelve mice were immunized with each construct three times at 4 week intervals, using 50 µg of plasmid into each set of quadriceps muscles for a total of 100 µg. No adjuvants were used (Bharadwaj et al., 2002).
Challenge Protocol:
Challenged the mice in the challenge replicate with 5 ID50 of SN77734 by the i.m. route 2 weeks after the third vaccination, a dose that corresponds roughly to 50–200 focus-forming units (Bharadwaj et al., 2002).
Efficacy:
Study used a deer mouse infection model to test the protective efficacy of genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological responses in BALB/c mice. Protective epitopes were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus G1 glycoprotein antigen; the nucleocapsid gene also was protective (Bharadwaj et al., 2002).
11. Sin Nombre virus DNA vaccine encoding SNVsSgp1 (NP)
Description:
Cloned the G1 and G2 glycoprotein genes of SN virus strain CC107 into the CMV expression vector pCMVi (-H3) UBs (Bharadwaj et al., 1999 ). The M segment fragments 3' of the first fragment were prepared in a similar manner, such that each expression construct shared 100 nt of sequence at the 5' end with the 3' end of the fragment that preceded it. The coordinates of each of the ten glycoprotein fragments, designated M-CMV-A thorough -I. We also cloned the entire SN virus N gene in a single fragment in a separate expression construct. The same viral cDNA fragments were cloned into bacterial expression vectors to allow bacterial synthesis of the cognate antigens as fusion proteins (Bharadwaj et al., 2002).
Vaccination Protocol:
Five to twelve mice were immunized with each plasmid DNA construct three times at 4 week intervals, using 50 µg of plasmid into each set of quadriceps muscles for a total of 100 µg. No adjuvants were used (Bharadwaj et al., 2002).
Challenge Protocol:
Challenged the mice in the challenge replicate with 5 ID50 of SN77734 by the i.m. route 2 weeks after the third vaccination, a dose that corresponds roughly to 50–200 focus-forming units (Bharadwaj et al., 2002).
Efficacy:
Study used a deer mouse infection model to test the protective efficacy of genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological responses in BALB/c mice. Protective epitopes were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus G1 glycoprotein antigen; the nucleocapsid gene (SNVsSgp1) also was protective (Bharadwaj et al., 2002).
Vaccination Protocol:
To asses the immunogenicity and protective capacity of the expressed rN proteins, 4- to 10-week-old bank voles, derived from a PUUV-free colony established with animals captured in Sweden, were immunized with purified PUUV, TOPV, ANDV, or DOBV rN or DHFR control protein. Bank voles were immunized three times with 50 μg of protein at intervals of 3 weeks. The animals were injected with protein emulsified in Freund's complete adjuvant, incomplete Freund's adjuvant, and phosphate-buffered saline (PBS), respectively (de et al., 2002).
Challenge Protocol:
Bank voles were challenged subcutaneously 2 weeks after the last immunization with approximately 20 50% infective doses of wild-type PUUV (strain Kazan) (de et al., 2002).
Efficacy:
To investigate the ability of recombinant N (rN, nucleocapsid proteins) from different hantaviruses to elicit cross-protection, we immunized bank voles with rN from Puumala (PUUV), Topografov (TOPV), Andes (ANDV), and Dobrava (DOBV) viruses and subsequently challenged them with PUUV. All animals immunized with PUUV and TOPV rN were completely protected (de et al., 2002).
Description:
Dob-N rNp was emulsified in 2% Alhydrogel (Accurate Chemical & Scientific Corp., Westbury, N.Y., USA). The rNp P40-Dob-N, P40-Dob118 and P40p-Dob118 were administered in sterile PBS (Maes et al., 2006).
d. Preparation
DOBV RNA (strain DOB-90/5) was extracted from infected Vero E6 cells (CRL 1586; ATCC, USA). The genomic RNA of DOBV was reverse-transcribed and PCR amplified in order to generate the entire S-segment, using following oligonucleotide primers: 5'-GCGAATTCGCAACACTAGAGGAACTCCAAAAGG-3' and 5'-CGAAGCTTAGTGGTGGTGGTGGTGGTGAAGTTTGAGCGGCTCC-3'. The amino-terminal part encoding the first 118 amino acids was generated using 5'-CTGGCGCCTAACCGACGTGGTGGTGGTGGTGGTGATTCGAAGC-3' as reverse primer. In all constructs, a histidine (His) tag was introduced at the C-terminal end. PCR fragments were cloned in plasmids pTEX(rP40) or pTEXmp18 respectively with or without the inclusion of the P40 sequence in the construct. Following transformation of the E. coli ICONE 200 strain, the recombinant proteins were produced as intracellular inclusion bodies, recovered, and renatured. The recombinant proteins were purifi ed by metal chelate affinity chromatography using a HisTrap kit (Pharmacia, Puurs, Belgium). Using this protocol, four DOBV rNp constructs were expressed and purified. The complete nucleocapsid protein of DOBV was expressed with or without the addition of the rP40 protein (constructs P40-Dob-N and Dob-N). The amino-terminal part of the DOBV nucleocapsid protein was expressed with the addition of the rP40 protein (construct P40-Dob118) or with the addition of only the periplasmic part of the rP40 protein (construct P40p-Dob118) (Maes et al., 2006).
e. Virulence
Not virulent
f.
Mouse Response
Host Strain:
NMRI mice (Elevage Janvier, Le Genest Saint Isle, France)
Vaccination Protocol:
Groups of ten 6-week-old NMRI mice (Elevage Janvier, Le Genest Saint Isle, France) were immunized three times subcutaneously with three different concentrations (0.2, 2 and 10 ug) of rNp with intervals of 2 weeks. The animals were injected with Dob-N rNp emulsifi ed in 2% Alhydrogel (Accurate Chemical & Scientific Corp., Westbury, N.Y., USA). The rNp P40-Dob-N, P40-Dob118 and P40p-Dob118 were administered in sterile PBS. Blood was drawn 14 days after each immunization (Maes et al., 2006).
Persistence:
N/A
Side Effects:
None.
Challenge Protocol:
Groups of 10 NMRI mice were immunized three times subcutaneously with 10 ug of the different constructs with intervals of 2 weeks, and were challenged intraperitoneal with DOBV 2 weeks after the last immunization. Three weeks later, all mice were sacrificed and serum samples were collected. Mice immunized three times subcutaneously with 10 ug of rP40 were used as a control group (Maes et al., 2006).
Efficacy:
All recombinant proteins were found to be highly immunogenic after three immunizations of rNp. The immunizations resulted in the induction of a strong Np-specific IgG response with a predominance of IgG1 over IgG2b and IgG2a, suggesting a mixed Th1/Th2 cell involvement. A specific IgG3 response could not be detected. Mice immunized with recombinant DOBV rNp without rP40 showed lower nucleocapsid-specific antibody responses in comparison with the rP40-conjugated constructs, but all mice were found to be protected against DOBV challenge. The results indicate that the rNp constructs coupled to rP40, represent promising vaccine candidates (Maes et al., 2006).
V. References
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